1. Field of the Invention
This invention relates to a novel heterobifunctional composition useful in immobilizing reagents on plastic surfaces. More specifically this invention comprises a molecule with hydrophobic regions that can intercalate into plastic and hydrophobic reactive groups that can covalently attach to other molecules. When contacted with plastic these novel reagents can, in a single rapid step, produce a reactive surface capable of binding other reagents.
2. Background Information
The practice of biotechnology, and particularly diagnostics, has increased the demand for products requiring immobilized reagents. "Reagents" includes proteins, nucleic acids, cells, drugs, and small molecule haptens. Substrates are insoluble matrices for immobilzation and can be plastic, glass, silica, carbon, cellulose, or other materials. Plastics are particularly useful substrates as they can be formed into a variety of shapes such as cups, discs, dipsticks, spheres, fibers, tubes, membranes, and particles. Plastics are used as stirface coats. Additionally, plastics have a high degree of biocompatability, and may be produced of materials having excellent optical properties. Typical plastics useful as substrates include polypropylene, polystyrene, polyethylene, polyvinyl chloride, polysulfone, polycarbonate, cellulose acetate and others. Plastics of styrene, vinyl chloride and carbonate are widely used when optical properties are a consideration.
Plastics are often used directly as substrates for direct immobilization of macromolecules. Polystyrene and polyvinyl chloride will anchor large molecules by electrostatic attraction. However, small molecules require attachment to larger "carrier" molecules before being bound to the plastic. Also, poor binding to most plastics limits the use of adsorption immoblization to high surface area systems. For instance, polystyrene latex particles can immobilize far more protein molecules per gram of plastic than molded polystyrene products.
Modification of the plastic surface has been used to increase the electrostatic interaction and increase the binding of some reagents. Electrostatic interactions alone will immobilize only a limited number of reagents, and detergents introduced in the system can cause reagent loss.
Reagent molecules are typically immobilized on a substrate by way of a linker molecule. Homobifuctional and heterobifunctional compounds have been devised to link a group present on the reagent to a group present on the substrate. As examples, disuccimidyl suberate and glutaraldehyde are homobifunctional compounds that can covalently bridge an amine group on a reagent molecule to an amine group present on a substrate, such as aminopolystyrene. Additionally, some plastics, such as methyl methacrylate and polyethylvinylacetate, have been developed to bear hydroxyls that can be convened to reactive intermediates. Reactive groups that can be provided include epoxides, hydroxysuccinimide esters, aldehydes, nitrophenyl chloroformate, activated thioIs, trityl, tresyl chloride, or other means for reacting free amines, hydroxides or sulfhydryls.
Reagents can also be hydrophilic pigments. In some industries the application of an inert topcoat to plastic substrates is needed for coloring or improved wear characteristics.
Modifications of the plastic surfaces to bear amines, hydroxyls, and sulfhydryls that can be crosslinked or otherwise modified often results in undesirable characteristics, particularly opacity or decrease structural integrity.
One system that has become available involves incorporation of a methyl imine function. This product requires the end user to convert the methyl imine functionality to a reactive group by addition of crosslinkers (NUNC, Naperville, Ill.). Another system treats plastic with a copolymer of phenylalanine and lysine amino acids to provide a support for a crosslinker (U.S. Pat. No. 4,657,873; Gadow, et al.). Gadow et al. is typical of the other prior attempts at forming reactive surfaces in that binding of a reagent requires several steps and usually entails crosslinking a nucleophile on the reagent molecule with a nucleophile on the plate.
Bienarz et al. (U.S. Pat. No. 5,002,883) also uses an amine bearing surface in combination with a "bridging" molecule to crosslink a reagent molecule to a plastic surface. As does Tetsuo et al. (UK Patent number GB2184 127A) which specifically requires hydrophilic functional groups on the surface prior to forming a bond between the reagent and the surface. Packard et al. (U.S. Pat. No. 4,889,916) has a similar requirement for two functional groups to be crosslinked, however, in the case of Packard the reaction is between sulfhydryl groups on both the substrate and the reagent molecule.
The technology of means et al. (U.S. Pat. No. 4,808,530) produces reagent bearing surfaces by convening hydrophilic groups on proteins to hydrophobic moieties. When the derivatized proteins are contacted to unmodified plastics the protein is bound by nonspecific adsorption to the surface.
All of the above technologies require a plurality of steps to modify the surface and then crosslink the reagent molecule of interest, or as in the case of Means et al., to modify the protein itself for attachment. Bieniarz et al. describe their derivatization process as requiring several steps over several hours. Typical procedures involve a one hour pretreatment of a prederivatized aminopolystyrene bead, followed by one hour derivatization with several clean up steps, the final step of adding reagent member required overnight incubation. Likewise, Gadow et al. describes a first derivatization step involving heating and mixing, followed by agitation for 30 minutes at room temperature, followed by a 24 hour incubation. At this stage the technology still is incapable of protein binding. The treated plastic resin must be activated for an additional 30 minutes with glutaraldehyde, the actual crosslinking reagent, and washed prior to protein binding.
Means et al. stipulates protein modification prior to binding to a surface. The proteins were modified and purified over the course of several hours, and plastic surfaces were contacted with the protein for an additional several hours. Packard et al. describes labeling of protein species using a heterofunctional crosslinker. As in Means et al. the Packard technology involves several steps to modify a protein surface, again requiring several hours and extensive purification.
Tetsuo et al. specifies modifying both the protein and the plastic surface. Introduction of thiol groups into proteins required 1 hour plus gel filtration cleanup. Activation of a plastic support required several sequential steps over several hours, plus removal of the reactants. Immobilization of derivatized protein required an additional 24 hours plus cleanup.
Clearly, there is a need for a simple rapid agent tbr producing activated surfaces capable of binding reagents. In this application we describe a chemical agent capable of producing an activated surface in only a single step. The activated surface is then capable of binding an unmodified reagent without any additional process steps.